Métodos moleculares na identificação, caracterização e detecção de Phytophora cambivora (Petri) Buisman e Phytophora cinnamomi Rands associadas com a doença da tinta do castanheiro.
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2004
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Um total de 49 isolados de Phytophthora spp. associados com a doença da tinta do castanheiro foram obtidos, pelos métodos clássicos de isolamento, em diferentes situações de crescimento do castanheiro na região Norte de Portugal: viveiros, plantações jovens, soutos adultos e do solo. Os isolados de Phytophthora foram identificados por PCR-RFLP da região ITS-rDNA amplificada pelos “primers” universais ITS6 (Cooke & Duncan, 1997) e ITS4 (White et al., 1990). A restrição enzimática do fragmento amplificado foi realizada com as enzimas Alu I, Msp I e Taq I cujo perfil de restrição permite a identificação ao nível da espécie no género Phytophthora. A população de Phytophthora associada com a doença da tinta foi caracterizada quanto ao tipo de compatibilidade sexual, polimorfismo RAPD e sensibilidade ao metalaxil.
P. cinnamomi é a espécie predominante em viveiro, plantações jovens, soutos adultos e no solo junto das plantas mortas. Apenas quatro isolados foram identificados como P .cambivora e dois deles foram obtidos em plantas de castanheiro com sintomas característicos da doença no colo da planta. Todos os isolados (P. cambivora e P. cinnamomi) são A2 e 9 % dos isolados de P. cinnamomi evidenciaram sensibilidade reduzida ao metalaxil. A caracterização RAPD permitiu a separação das espécies de Phytophthora associadas à doença da tinta embora os primers “10-mer” utilizados não tivessem possibilitado a caracterização dos isolados de P. cinnamomi. O estudo RAPD permitiu, no entanto, identificar um fragmento de amplificação característico de P. cinnamomi. O fragmento foi purificado clonado e sequenciado (SCAR) e desenharam-se primers específicos (Cin1, Cin2 e Cin3) para a detecção e identificação de P. cinnamomi por PCR. Os primers utilizados na combinação Cin2/Cin1 e Cin3/Cin1 proporcionam um produto de amplificação PCR com aproximadamente 650 pb. Com esta metodologia de diagnóstico não se detectaram reacções cruzadas com as espécies de P. cactorum, P. capsici, P. cinnamomi var. parvispora, P. citricola, P. cryptogea,P. nicotianae, P. quercina, P. syringae e ainda com Cryphonectria parasitica, Pythium sp. E Castanea sativa. O método PCR-Diagnóstico foi aplicado com sucesso, em raízes de castanheiro, que cresceram em substrato inoculado com P. cinnamomi, em tecidos vegetais (folhas de castanheiro) e na água da técnica armadilha de detecção de Phytophthora no solo.
A total of 49 isolates of Phytophthora spp. associated with ink disease of chestnut were obtained by classical methods from diverse chestnut growing regions of the north of Portugal: nurseries, young and old chestnut groves, and also in the soil near dead plants of sweet chestnuts. Phytophthora isolates were identified by PCR-RFLP of ITS-rDNA amplified by the universal primer pair ITS6 (Cooke & Duncan, 1997) and ITS4 (White et al., 1990). Restriction digestions of the amplified DNA product were performed with restriction enzymes Alu I, Msp I, and Taq I, which allowed individual species identification. The population of Phytophthora was characterised in terms of mating type, metalaxil resistance and RAPD polymorphisms. P. cinnamomi is the most prevalent species in nurseries, orchards and in soil of chestnut groves. Only four isolates were identified as P. cambivora and two of them were obtained from typical rot crown of chestnut. All the isolates (P. cambivora and P. cinnamomi) were mating type A2 and 9 % of P. cinnamomi were metalaxil tolerant. Although RAPD polymorphisms allowed species differentiation the “10-mer” primers used were not suitable for P. cinnamomi characterization. The RAPD analyses allowed, however, the identification of a fragment characteristic of P. cinnamomi. The fragment was purified, cloned and sequenced (SCAR) and then species-specific primers were designed (Cin1, Cin2, and Cin3) for detection and identification of P. cinnamomi by PCR. Each primer pairs Cin2/Cin1 and Cin3/Cin1 produced an amplicon of approximately 650 bp from all tested P. cinnamomi isolates. Both primer pairs revealed no undesirable cross-reaction with a diverse range of Phytophthora species (P. cactorum, P. capsici, P. cinnamomi var parvispora, P. citricola, P. cryptogea,P. nicotianae, P. quercina, P. syringae), Cryphonectria parasitica, Pythium sp., and Castanea sativa. The PCR-Diagnostic assay based on the species-specific primers was successfully used to detect P. cinnamomi in roots of chestnut plants, which were grown in artificially infected substrate with this pathogen, in bait tissue (chestnut leaves) and in the water of the same bait test.
A total of 49 isolates of Phytophthora spp. associated with ink disease of chestnut were obtained by classical methods from diverse chestnut growing regions of the north of Portugal: nurseries, young and old chestnut groves, and also in the soil near dead plants of sweet chestnuts. Phytophthora isolates were identified by PCR-RFLP of ITS-rDNA amplified by the universal primer pair ITS6 (Cooke & Duncan, 1997) and ITS4 (White et al., 1990). Restriction digestions of the amplified DNA product were performed with restriction enzymes Alu I, Msp I, and Taq I, which allowed individual species identification. The population of Phytophthora was characterised in terms of mating type, metalaxil resistance and RAPD polymorphisms. P. cinnamomi is the most prevalent species in nurseries, orchards and in soil of chestnut groves. Only four isolates were identified as P. cambivora and two of them were obtained from typical rot crown of chestnut. All the isolates (P. cambivora and P. cinnamomi) were mating type A2 and 9 % of P. cinnamomi were metalaxil tolerant. Although RAPD polymorphisms allowed species differentiation the “10-mer” primers used were not suitable for P. cinnamomi characterization. The RAPD analyses allowed, however, the identification of a fragment characteristic of P. cinnamomi. The fragment was purified, cloned and sequenced (SCAR) and then species-specific primers were designed (Cin1, Cin2, and Cin3) for detection and identification of P. cinnamomi by PCR. Each primer pairs Cin2/Cin1 and Cin3/Cin1 produced an amplicon of approximately 650 bp from all tested P. cinnamomi isolates. Both primer pairs revealed no undesirable cross-reaction with a diverse range of Phytophthora species (P. cactorum, P. capsici, P. cinnamomi var parvispora, P. citricola, P. cryptogea,P. nicotianae, P. quercina, P. syringae), Cryphonectria parasitica, Pythium sp., and Castanea sativa. The PCR-Diagnostic assay based on the species-specific primers was successfully used to detect P. cinnamomi in roots of chestnut plants, which were grown in artificially infected substrate with this pathogen, in bait tissue (chestnut leaves) and in the water of the same bait test.
Descrição
Tese de Doutoramento em Ciências Agronómicas / Protecção de Plantas
Palavras-chave
Castanea , Phytophora cinnamomi , Phytophora cambivora , Doença da tinta do castanheiro , Ink disease of chestnut , PCR-Diagnostic